GENE EXPRESSION PROFILES AND PRODUCTS FOR THE DIAGNOSIS AND PROGNOSIS OF POSTINJURY SYNOVITIS AND OSTEOARTHRITIS
Associations between inflammation and pain/function scores were tested by univariate and multivariate analyses. Gene expression was analyzed by microarray and real-time PCR comparing patients with and without synovial inflammation. Synovitis was present in 43% of patients presenting for arthroscopic menisectomy. Inflammation was associated with pre-operative Lysholm scores, independent of age, gender, and BMI. Synovial RNA microarray analysis revealed 260 genes differently expressed ≧2-fold between patients with and without synovitis. A chemokine signature identified in the “inflammatory” biopsies was confirmed by real-time PCR. In conclusion, in patients presenting for arthroscopic menisectomy, synovitis is associated with symptoms. Comparison of expression patterns revealed enrichment of chemokines associated with cellular recruitment and activation in patients with synovitis. These chemokines may represent targets for therapeutic intervention to reduce inflammatory symptoms in patients with meniscal injury.
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This patent application is a continuation-in-part of copending International Application No. PCT/US2011/051773, filed Sep. 15, 2011, which claims priority to U.S. provisional application Nos. 61/383,110, filed Sep. 15, 2010, and 61/383,594 filed Sep. 16, 2010. The disclosures set forth in the referenced applications are incorporated herein by reference in their entireties, including all information as originally submitted to the United States Patent and Trademark Office.
SEQUENCE LISTINGThe instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 15, 2011, is named 700726_SEQ_ST25.txt and is 259,333 bytes in size.
BACKGROUNDJoint injury predisposes individuals to develop osteoarthritis (OA). Among the most common knee joint injuries associated with increased OA risk are meniscal injuries. Recent longitudinal data from the Multicenter Osteoarthritis Study indicate that meniscal damage is associated with a 6-fold increased risk (OR 5.7, 95% CI 3.4-9.4) of developing radiographically visible OA changes. Furthermore, in patients with established OA, meniscal damage is associated with increased risk of progression. Anatomic patterns of meniscal tear are often utilized to discriminate between traumatic and degenerative meniscal pathology; traumatic tears occurring in an otherwise normal meniscus are reported to present with longitudinal (sometimes “bucket-handle” type tears) or radial orientations, while horizontal, flap or complex tears and maceration are interpreted as degenerative tears, i.e. those occurring in a meniscus structurally weakened by degenerative change. Both patterns of meniscal alteration are associated with elevated risk of OA, but the risk associated with degenerative-type tears appears to be higher. Although biomechanical factors likely play a role in the structural changes in both patterns of meniscal pathology, the cellular and molecular processes that lead to increased risk of OA are not understood. Furthermore, these injuries are often asymptomatic, and factors that contribute to symptoms such as pain have not been defined.
In patients with OA, inflammation is one factor associated with risk of both progression of cartilage loss and symptoms. Inflammation in OA joints manifests as synovial membrane (SM) mononuclear cell infiltration observed in both early and late stages of disease. However, it is not clear whether inflammation pre-dates or is a consequence of early OA development. Roemer and colleagues recently noted an association between meniscal damage and synovial effusion on MRI, but the cellular and molecular nature of this inflammation was not clear. Pessler et al. noted a mild synovitis with histologic features similar to OA in a heterogeneous group of patients with “orthopedic arthropathies”, including some with meniscal tears. However, the prevalence of inflammation in patients with meniscal injuries in the absence of preexistent OA has not been established.
Predictive factors of OA risk joint injuries are needed to guide clinical treatment.
SUMMARYA gene expression profile is disclosed with values for gene products that are differentially expressed in knee injury patients with synovial inflammation compared to patients without synovial inflammation. In an embodiment, the profile includes the genes of Annex Table 2. Gene products include mRNA, usually measured by PCR methods disclosed herein, and proteins, measured according to methods known in the art (also see herein).
In another embodiment, the profile includes the genes of Table 3. The gene expression profile wherein cytokine (chemokine) gene expression was used, was positively associated with Lysholm scores, a knee-specific metric of symptoms, and functional disability.
In particular, expression of chemokine IL8, CCL5, CCL19 and CCR7 was associated with synovial inflammation.
The gene expression profiles that are differentially expressed in knee injury patients with and without synovial inflammation are useful to identify a patient with knee symptoms associated with synovial inflammation. To determine the gene expression profile from a biological sample of the patient, the methods disclosed herein result in vectors of expression values.
The profile of the patent is compared to profiles obtained from patients with knee injuries who had synovial inflammation, and those who did not, to determine to which group the patient most likely belongs. If synovial inflammation contributes to knee symptoms of the patient, clinical treatment will address the inflammation.
A method to target genes in the expression profile of a patient, includes the steps of:
(a) determining which genes in the patient's genetic profile show the greatest association with synovial inflammation; and
(b) targeting those genes for developing therapies.
A method of treatment associated with knee injuries in a patient includes treating the patient by interacting with the targets to alleviate their effects.
The targets may be chemokines, in which case inflammation will be alleviated.
To improve clinical outcomes after arthroscopic and post joint trauma in a patient:
(a) determine the chemokine signature of the patient; and
(b) select a treatment based on the target genes that are in the chemokine signature.
Gene expression profiles were used to identify knee injury patients with inflammation. There was an initial traumatic meniscal tear patient cohort, and a repository patient cohort. Microarray analysis of synovial RNA initially revealed that 260 genes (Annex—Table 2) were differentially expressed between patients with and without inflammation. Chemokine and chemokine receptors were among the most upregulated transcripts in biopsies with inflammation. Inflammation is defined herein as perivascular mononuclear cell aggregates, which are largely composed of lymphocytes
Classification of patients by identification of genes associated with synovial inflammation is useful to determine appropriate control of clinical symptoms. Markers of early symptomatic disease and prognosis are based on an association between synovial inflammation and clinical symptoms in patients with meniscal degeneration, irrespective of the presence of underlying cartilage degeneration.
Because of the association with inflammation, therapeutic strategies are contemplated. Targeted, intra-articular injection therapies (i.e. corticosteroids and hyaluronan-derivatives) reduce symptoms in both OA and joint injury. IA corticosteroids in particular act as broad-spectrum anti-inflammatory agents. Therapeutics may be targeted to block chemokine activity and/or production in joints to attenuate recruitment and activation of inflammatory cells. These therapeutics are delivered either systemically in the case of patients with multi-joint OA, or locally by intra-articular injection in the case of patients with disease or traumatic injury limited to a single joint. In the case of local injection, systems for slow or sustained release are employed to deliver a more sustained therapeutic response to reduce inflammatory symptoms.
Some reports indicated that synovitis is related to OA symptoms and progression of the condition. Synovial inflammation and effusions also occur with meniscal injuries, even in patients without radiographic evidence of OA. However, cellular and molecular characteristics of synovial reactions associated with meniscal damage have not been reported. The prevalence and the molecular features of synovial inflammation were determined in patients who were (i) without preexistent radiographic features of OA, and who were (ii) undergoing arthroscopic meniscectomy for clinically-documented traumatic knee injury associated with MRI evidence of meniscal pathology. A specific goal was to determine whether synovial inflammation correlated with clinical symptoms and whether gene expression profiles could predict synovial inflammation.
A histologic scoring system to grade inflammation was validated using independent evaluators, and comparisons were made with previously characterized synovial tissue from patients with early or late stage OA.
Appearance of cellular infiltrates was similar, but inflammation was less prevalent and extensive in meniscectomy patients. Unexpectedly, there was not preferential localization of inflammation on the side of the meniscal tear. Instead, inflammation was most prevalent in the suprapatellar location (in 43% of patients), indicating that synovial inflammation occurs globally within the joint at sites distant from the injury. Certain sites within the joint may be uniquely sensitive to effects of proinflammatory factors produced in response to meniscal injury.
Another question was whether inflammation was associated with preoperative joint symptoms and dysfunction. When stratified according to presence or absence of suprapatellar synovial inflammation, Lysholm scores were significantly lower (p<0.05) in patients with synovial inflammation, indicating a higher degree of knee-related symptoms and dysfunction. No differences in SF-12® or VAS pain scores were observed. Unlike these two scales, the Lysholm score is a knee-specific metric of symptoms (pain, swelling, limp, locking and instability) and functional disability (stair-climbing, squatting and use of supports). It is scored on a scale of 0-100 (100=best), with pain and instability-related symptoms having most weight (25 points each). In contrast, the VAS scale only measures pain, and the SF-12® health survey measures physical, social and mental health. Neither is specific for knee-related issues. The unique association of inflammation with Lysholm scores and not VAS pain scores suggests that symptoms other than pain (e.g., instability, swelling) captured by the Lysholm scale account for this difference. The weighting of the scale may also contribute to these observations.
Patient characteristics [(age, body mass index (BMI), degree of cartilage abnormality, and time elapsed between injury and surgery) were analyzed in the stratified data. [Table 1(a)] Age and BMI are known risk factors for OA. In this cohort, older patients were more likely to demonstrate synovial inflammation, but BMI did not differ with inflammation score. It was expected that infiltration of cells would increase with time elapsed between injury and surgery, but this did not appear to be true. Patients with synovial inflammation tended to have shorter time intervals between injury and surgery. Possibly increased inflammatory symptoms prompt earlier intervention. Multivariate analysis indicated that the association between inflammation and Lysholm scores is independent of age, BMI, and interval between injury and surgery.
The population examined is one in which an identifiable injury precipitated symptoms, and who had tears that did not involve the vascular portion of the meniscus. Also, despite a clear history of trauma, most patients exhibited complex meniscal lesions upon arthroscopic examination. Although patients with clinical or radiographic signs of OA were excluded, most patients demonstrated grade 1-4 Outerbridge cartilage lesions suggesting this population is enriched for patients with pre-radiographic disease. These observations indicate the presence of an early degenerative process occurring within the joint of the majority of these patients, given the known association between pre-existing OA and a complex pattern of meniscal pathology. Because synovial inflammation is associated with symptoms in patients with established OA, a question was whether inflammation was related to the degree of underlying cartilage abnormality as a sign of early OA. There was a trend towards greater inflammation in patients with cartilage abnormalities, but the multivariate model demonstrated that the association between inflammation and Lysholm scores was independent of degree of cartilage abnormality. The finding of synovial inflammation in one of seven patients with normal cartilage suggests that in some cases of meniscal injury, inflammation may pre-date cartilage changes.
To obtain insight into molecular markers that contribute to synovial inflammation, a microarray analysis of synovial RNA was performed. Four biopsies from patients with inflammation (grade 1 or 2) and four without (grade 0) were compared. 260 genes (Annex Table 2) were differentially expressed between these two patient groups (≧2 fold change). Inflammatory pathway over-representation analysis of these differentially expressed genes revealed twenty-two “pathways” (transcripts which cluster into functional categories or molecular pathways) that were significantly enriched with corrected p values <0.05.
Seven clusters were embodied which included more than three gene products. Of these seven, a signature of chemokines and their receptors was the top up-regulated pathway in biopsies exhibiting inflammation. The six transcripts in this signature are shown in Table 2, with their respective fold-change and p-values. The other six pathways identified were “Primary Immunodeficiency” and “Hematopoietic cell lineage” composed of cell surface receptors and genes associated with infiltrating leukocyte populations (i.e. CD19, IL2RG, IL7R, CIITA, CD1D, CD2).
Three additional pathways included overlapping lists of cytokine receptor chains (IL2RB, IL2RG), an intra-cellular signaling molecule (JAK3), and cytolytic enzymes (GZMA, GZMB) expressed by T and NK cell populations, and related to IL-12 signalling. The seventh pathway, “Cytokine-cytokine receptor interactions,” was largely comprised of the same six chemokine/receptor transcripts identified in the chemokine signature. These seven pathways were condensed to the three listed in Table 3. In addition, a number of genes involved in B lymphocyte activity and signaling were identified in the differentially expressed gene set and are also shown in Table 3. For the purpose of the present analysis, chemokines were a focus because of their potential contribution to early events in lymphocyte accumulation in synovium.
The gene expression profiles suggest that the chemokine signature identifies a group of patients with synovial inflammation and knee symptoms. Given the role of these chemokines in recruitment of inflammatory cells, they may contribute to development of synovial inflammation in response to meniscal injury. Conclusions include:
-
- (a) inflammation is detectable in 43% of patients with degenerative menisci without radiographic OA; it is not more prevalent on the side of injury;
- (b) inflammation is associated with worse pain/dysfunction;
- (i) although usually associated with pre-existing OA changes in the cartilage, inflammation is occasionally detected in patients with normal cartilage after meniscal injury;
- (c) inflammation is associated with chemokine expression;
- (i) histologic inflammation, CCL19/CCR7, IL-8 and CCL5 levels are associated with Lysholm scores;
- (ii) chemokines may represent markers of early disease or have predictive value for persistent pain, worse surgical outcomes, progression of symptoms.
Expression of these genes (Table 3) within the synovium may promote recruitment of the inflammatory cellular infiltrate, so this gene set was selected for validation by real-time PCR.
Five genes were selected for validation by real-time PCR: IL-8, CCL5, CCR7, CCL19, and CCL21. With the exception of IL-8, these belong to the “C-C” chemokine gene family which generally influences recruitment of monocytes, lymphocytes and eosinophils. IL-8, a “C-X-C” chemokine, recruits neutrophils to sites of inflammation. Although first described as a T-lymphocyte recruitment factor, CCL5 (or RANTES) has pleiotropic effects on multiple leukocyte subsets. CCR7 is the cognate receptor for both CCL19 and CCL21, which are involved in T-lymphocyte and dendritic cell migration; interaction between these chemokines and their receptor mediates homing to secondary lymphoid tissues and appropriate migration of cells within lymphoid follicles. Analysis revealed increased IL-8, CCL5, CCR7 and CCL19 relative expression levels in biopsies with inflammation (
Expression of genes that make up the “chemokine signature” (Table 3: IL-8, CCL5, CCL19, CCL21, XCL1, CCR7, CXCR3, CXCR6) may be measured in clinical/biological fluids (synovial fluid from affected joint, peripheral blood, urine) obtained during office visits or surgical procedures by methods such as ELISA, Elispot, or a high-throughput techniques such as Luminex bead-based detection. Cells derived from synovial fluid or blood may potentially be analyzed by flow cytometry for the presence of the receptor CCR7, or cell-bound or intracellular chemokine production. Alternatively, transcripts of these chemokines and receptor may be measured in synovial tissue biopsies taken at the time of surgical intervention, or office-based needle biopsy of the affected joint (i.e. the suprapatellar pouch).
Utility of the Classification of Patients by Gene Expression ProfilesPost-Traumatic Knee Injury:
Diagnosis: (a) detection of co-existing early-stage (pre-radiographic) osteoarthritis that is associated with synovial inflammation guides clinical decision making in determining whether a patient is a good surgical candidate or not (b) detection of local, chronic inflammatory response in association with the injury guides choice of therapeutics (i.e. corticosteroids, hyaluronan injections, or future targeted therapeutics) used alone or in conjunction with surgical approaches. Prognosis: (a) determination of an individual patient's risk of sustained inflammatory symptoms post-surgery guides clinical follow-up and (b) determination of an individual patient's risk of more rapid progression to overt Osteoarthritis, guides both current clinical trial planning as well as future therapeutic/preventative interventions.
Patients with Unexplained Knee Pain:
Diagnosis: detection of early-stage (pre-radiographic) osteoarthritis that is associated with synovial inflammation guides appropriate treatment strategies. These tests have advantages in enhancing the predictive value of existing imaging techniques (i.e. MRI) to define patients at greater risk for inflammatory symptoms. Prognosis: determination of an individual patient's risk of future osteoarthritis, guides both current clinical treatment planning as well as future therapeutic/preventative interventions.
Patients with Known Osteoarthritis:
Diagnosis: detection of an associated chronic inflammatory response, guides treatment choices targeting inflammatory symptoms. Prognosis: determination of an individual patient's risk of more rapid progression of existing disease, guides clinical treatment planning as well as therapeutic/preventative interventions.
EXAMPLESExamples are provided for illustrative purposes and are not intended to limit the scope of the disclosure.
Example 1 The Post-Traumatic Meniscectomy CohortTraumatic and degenerative meniscal tears have different anatomic features and different proposed etiologies, yet both are associated with development or progression of osteoarthritis (OA). In established OA, synovitis is associated with pain and progression, but a relationship between synovitis and symptoms in isolated meniscal disease has not been reported. Synovial pathology in patients with traumatic meniscal injuries was characterized and the relationships between inflammation, meniscal and cartilage pathology, and symptoms were determined.
Thirty-three patients ([Table 1(a)]) without evidence of OA who were undergoing arthroscopic meniscectomy for meniscal injuries were recruited. Pain and function were assessed preoperatively; meniscal and cartilage abnormalities were documented at the time of surgery. Inflammation in synovial biopsies was scored and associations between inflammation and clinical outcomes determined. Microarray analysis of synovial tissue was performed and gene expression patterns in patients with or without inflammation compared.
Synovial inflammation was present in 43% of patients and was associated with worse pre-operative pain and function scores, independent of age, gender, or cartilage pathology. Microarray analysis and real-time PCR revealed a chemokine signature in synovial biopsies with increased inflammation scores.
In patients with traumatic meniscal injury undergoing arthroscopic meniscectomy without clinical or radiographic evidence of OA, synovial inflammation occurs frequently and is associated with increased pain and dysfunction. Synovia with increased inflammation scores exhibit a unique chemokine signature. Chemokines may contribute to the development of synovial inflammation in patients with meniscal pathology; they also represent potential therapeutic targets for reducing inflammatory symptoms.
Patients:
The study was approved by the Institutional Review Board (IRB) of the New England Baptist Hospital, and all patients gave written, informed consent. Patients aged 18 to 60 years who suffered a traumatic knee injury and were scheduled for arthroscopic partial meniscectomy for treatment of symptomatic meniscal tears were recruited from the Department of Orthopedic Surgery at New England Baptist Hospital. The inclusion criterion was patient recall of an injury to the knee which initiated their symptoms and which occurred within six months of initial presentation, and a meniscal tear identified on pre-operative MRI and considered to be the cause of the symptoms. Exclusions were (i) those with known inflammatory arthritis, and clinical or radiographic evidence of OA (osteophytes or joint space narrowing), and (ii) patients with meniscal tears affecting the vascular portion of the meniscus thought to be amenable to surgical repair rather than resection. The latter was done to increase the homogeneity of the patient population.
Outcome Scores:
The Short form-12 (SF-12®) health surveys, Lysholm questionnaires, and visual analog pain scales (VAS) were administered pre-operatively. The Lysholm questionnaire is a knee-specific instrument for measuring symptoms (pain, swelling, limp, locking and instability) and functional disability (stair-climbing, squatting and use of supports) on a single scale (0-100). Originally developed to assess responses to ligamentous repairs, this score has been validated in patients undergoing meniscal procedures. In contrast, SF-12® is a generic health survey capturing information on general physical and emotional well-being.
Assessment of Meniscus and Cartilage Integrity:
Surgical reports were available for 28 patients, and were reviewed to determine the anatomic pattern of meniscal pathology (degenerative vs. traumatic). The degree of cartilage damage was assessed intra-operatively using the Outerbridge scoring system: 0=normal articular cartilage, 1=superficial softening, 2=superficial fissuring or fibrillation involving <1.25 cm area, 3=fibrillation or fissuring with >1.25 cm area, 4=full-thickness cartilage wear with exposed subchondral bone.
Synovial Tissue Collection and Preparation:
Tissue from patients undergoing meniscectomy was obtained from three defined locations: suprapatellar pouch, medial and lateral gutters. Tissue biopsies were formalin-fixed and paraffin-embedded before sectioning and H&E staining.
Histologic Assessment of Synovial Inflammation:
To standardize evaluations, only sections containing a clearly recognizable synovial lining layer with underlying vascularized subintima were analyzed. Comparisons were made to suprapatellar biopsy specimens from patients with known knee OA, both early and end-stage. To further standardize, inflammation was evaluated at low-power (10× objective). As there are no published reports on synovial infiltrates in patients with meniscal injury only, inflammation was graded based on perivascular mononuclear cell infiltration in the synovial membrane from OA patients as follows: grade 0=none, grade 1=mild (0-1 perivascular aggregates per low-power field); grade 2=moderate (>1 perivascular aggregate per low power field with or without focal interstitial infiltration); grade 3=marked aggregates (both perivascular and interstitial). To evaluate inter- and intra-reader reliability, subsets of specimens were scored by two independent readers (E.D., C.R.S.) and were re-read by one blinded reader (E.D.).
Synovial Gene Expression Microarray Analysis:
Total RNA was extracted from homogenized SM samples using PerfectPure® RNA Fibrous Tissue kits (5Prime Inc., Gaithersburg, Md.). All RNA was DNAse-treated, oligo-dT primed, and cDNA synthesized with SuperScript III® Reverse Transcriptase (Invitrogen Life Technologies, Carlsbad, Calif.). RNA integrity was determined by electrophoresis on a microfluidics-based platform (Agilent Technologies, Santa Clara, Calif.). Eight synovial biopsies were chosen for microarray analysis, four each from meniscectomy patients with synovial inflammation (grade 1 or 2) or without synovial inflammation (grade 0) where synovial inflammation was identified histologically. RNA was hybridized to Affymetrix human U133 plus 2.0 chips at the Cornell University Weill College of Medicine Core Facility. Data were analyzed using Genespring 10.0 software (Agilent Technologies) as follows. Data were transformed using the RMA algorithm with baseline transformation to the median of all arrays. Probesets were filtered by expression (20-100%), with the requirement that probes be present in at least 4 of the 8 arrays. An unpaired t test was done on the filtered data. 3030 probesets were differentially expressed in synovial inflammation samples (p<0.05); 260 were differentially expressed with a >2-fold difference. Pathway over-representation analysis was done utilizing algorithms available via the Innate DB database (http://www.innatedb.ca/index.jsp) Innate DB is a database of genes, proteins, interactions and signaling responses involved in the mammalian innate immune response. (Lynn et al. Molecular Systems Biology 2008:4:218) Targets were then chosen for validation by real-time qPCR.
Quantitative PCR Analysis:
mRNA levels of four chemokines and one chemokine receptor identified by microarray pathway analysis (IL-8, CCR7, CCL19, CCL21 and CCL5) were measured by real-time PCR using specific primers and iQ Sybr-Green Supermix (BioRad, Hercules, Calif.). Primers spanned introns and yielded a single product. After normalizing Ct values to GAPDH, expression levels were calculated relative to the mean of specimens without inflammation.
Statistical Analysis:
Inter- and intra-reader reliability of inflammation scores is reported as a weighted kappa statistic. Given the small sample size and some irregularly distributed variables, nonparametric tests were used. Between-group differences were evaluated with Mann-Whitney t-tests, and Spearman's correlation coefficients were calculated using Prism 5.0 software (GraphPad, Inc., San Diego, Calif.). Multiple linear regression analysis was performed to examine the association between synovial inflammatory score and baseline Lysholm scores. Age, gender, BMI and time between injury and surgery were included as independent covariates.
Patient Characteristics:
Thirty-three patients were recruited. All patients reported a history of traumatic knee injury which precipitated their symptoms and all underwent arthroscopic partial meniscectomy; patients undergoing meniscal repairs were excluded, as were patients with evidence of OA on pre-operative knee x-rays (i.e. Kellgren-Lawrence scores>0). Demographics of these patients (age, BMI, gender) are presented in Table 1(a). The median interval between knee injury and surgery was 14.8 weeks (range 1-42 weeks). Most (26, 82%) had medial meniscal tears; six had lateral tears; one both medial and lateral tears. Surgical reports were available for 28 patients; twenty-five reports indicated the presence of complex tears, with horizontal cleavages and flap lesions and one described as macerated. Only two had isolated radial tears (one patient had both medial and lateral tears, one radial and one complex), and two were unrecorded. Using the Outerbridge scale to assess cartilage integrity, only seven patients (21%) scored zero (normal cartilage) in all compartments. The remainder had grade 1 (n=6), grade 2 (n=7), or grade 3 (n=7) lesions in one or more compartments, with 6 exhibiting focal, grade 4, chondral lesions but no diffuse full-thickness cartilage loss.
Histologic Assessment of Synovial Inflammation:
Biopsies of sufficient quality and quantity for evaluation were available from 28 patients. Inflammation was graded 0-3 based on perivascular mononuclear cell infiltration in H&E sections. Zero represents no inflammation; 3 marked inflammation. As there were no reports describing synovial infiltrates in this patient population, the scale used was based on perivascular mononuclear cell infiltration in OA patients. Hence, prevalence and extent of inflammation in the patients was compared to a group of 20 OA patients (6 with early knee OA, as defined previously; 14 with advanced stage OA undergoing joint replacement).
Table 1(a) shows demographics of these patients. Median Body Mass Index (BMI) was similar in meniscectomy and OA patients, but OA patients were older (medians, 64 vs. 48 years, p<0.0001) and more likely to be female (Fisher's exact test, p<0.05).
Reliability of Histologic Score:
To evaluate inter- and intra-rater reliability of inflammation scoring, 18 synovial specimens were scored by two independent readers (E.D., C.R.S.) and 8 were re-scored by one blinded reader (E.D.). Inter-rater and intra-reader weighted kappas were 0.87 and 1.0 respectively, indicating good reliability.
Prevalence and Anatomic Variation of Inflammatory Infiltrates:
Synovial tissue was obtained from three anatomic locations in the meniscectomy patients: the suprapatellar pouch (n=28), and the medial and lateral gutters (n=27 each).
Association of Inflammation with Patient Characteristics and Lysholm Scores:
Patients were stratified according to the presence (n=12, score 1 or 2) or absence (n=16, score 0) of suprapatellar inflammation. Lower Lysholm scores (indicating greater knee-related symptoms and disability) were observed in patients with synovial inflammation than in patients without inflammation (difference between means=−19.9, 95% CI −9.20 to −30.7, p=0.0008). No significant differences in SF-12® (−0.85, 1.08 to −2.79) or VAS pain scores (0.44, 2.27 to −1.40) were observed. Patients with synovial inflammation were significantly older (51.3±7.3 years vs. 40.2±11.6, p=0.007), and the interval between injury and surgery was significantly shorter (10.2±8.8 weeks vs. 18.5±11.5, p=0.047). Inflammatory infiltrates were observed in some patients presenting for surgery within a few weeks of their reported injury. Despite excluding patients with clinical or radiographic OA, 60% of patients had evidence of Outerbridge grade 1-3 cartilage degeneration and 18% (n=6) had discrete grade 4 chondral lesions noted intra-operatively. Although there was no significant difference in mean Outerbridge cartilage scores, there did appear to be a trend towards higher Outerbridge scores in patients with synovial inflammation (2.3±1.2 vs. 1.3±1.5, p=0.07). Only one of the seven patients with normal (grade 0) cartilage scores showed inflammation. Of six with focal grade 4 lesions, five were female, but otherwise they were not clearly distinguishable from the rest of the cohort, and Lysholm scores varied widely (40-90). Synovial biopsies were available for four: two exhibited synovial infiltrates (grade 1); two did not. There was no correlation between Outerbridge scores and Lysholm scores (r=0.03, p=0.86).
Multivariate Analysis:
Multiple linear regression analysis was performed to determine whether the relationship between synovial inflammation and Lysholm scores was independent of known OA risk factors and of the degree of underlying cartilage abnormality. Suprapatellar scores were analyzed because inflammation was most prevalent in this location. Age, gender, Outerbridge score, BMI and time between injury and surgery were included as independent covariates. Both inflammatory score (p=0.001, effect estimate −15.3±4.7 per point) and BMI (p=0.004, effect estimate −1.3±0.4 per kg/m2) were significantly associated with Lysholm score after adjusting for the above variables. Outerbridge score (p=0.69) and age (p=0.30) were not, after accounting for other variables.
Analysis of Synovial Gene Expression in Patients with or without Synovial Inflammation:
Histologic analysis was used to stratify biopsies according to the presence or absence of synovial inflammation for further analysis of gene expression using microarray technology. SM specimens from eight meniscectomy patients, four with and four without synovial inflammation were chosen for microarray analysis. The eight biopsies were from different patients; anatomic locations varied.
Validation of Chemokine Expression by Real-Time PCR:
mRNA levels of four chemokines and one chemokine receptor identified by microarray pathway analysis (IL-8, CCR7, CCL19, CCL21 and CCL5) were measured by real-time PCR. All available biopsies yielding sufficient cDNA quantities were utilized (36 samples representing 18 patients). Samples were stratified by inflammation score (±) and relative analyte expression levels were compared. Levels of IL-8 (
Association of Chemokine Expression with Baseline Lysholm Scores:
Associations between chemokine expression and clinical outcome scores were assessed by Spearman correlation. Only suprapatellar chemokine expression was analyzed. CCR7 (
To validate findings in the post-traumatic meniscectomy patients, synovial chemokine expression levels were measured in a separate cohort of patients: those enrolled in the Rush Knee Meniscal Injury and Osteoarthritis Repository (the “repository cohart”) the demographics of which patients are shown in Table 1b. This group included both patients with post-traumatic and idiopathic meniscal tears, as well as some patients with radiographic changes of established OA. The Knee Injury and Osteoarthritis Outcome Score (KOOS) surveys were administered at the time surgery was scheduled to evaluate functional status and symptomatology. The KOOS is a validated knee-specific patient questionnaire which evaluates both short-term and long-term symptoms and function in patients with knee injury and osteoarthritis. It consists of 5 scored subscales: Pain, Other symptoms, Activities of Daily Living (ADL), Function in Sports and Recreation, and Quality of Life (QOL).
The results in Table 4 demonstrate relationships between chemokine expression levels and KOOS scores in the repository cohort, that are consistent with observations in the post-traumatic meniscal injury cohort with no OA using the Lysholm score. These results suggest that the relationship between chemokine expression and symptoms may be applicable to a broader patient population encompassing all patients (post-traumatic and idiopathic) presenting for arthroscopic meniscal repair/resection with varying stages of early-intermediate radiographic OA. Similar findings in two cohorts of patients using two different, validated outcome scores (the KOOS and the Lysholm) further strengthen initial findings.
Relationships were demonstrated between symptoms and chemokine mRNA levels in synovial biopsies.
Higher chemokine mRNA expression levels measured in synovial biopsies from patients with post-traumatic meniscal tears were associated with worse Lysholm score indicating greater levels of knee symptoms and dysfunction.
Higher chemokine mRNA expression levels (including IL-8 and CCL19) measured in synovial biopsies were associated with knee symptoms and dysfunction measured by the Lysholm score. Data shown in
Data in
Patient subsets and disease states to which the synovitis markers are applicable include:
Joint-Specific Idiopathic Osteoarthritis: 1. Knee 2. Hip 3. Shoulder 4. Hand5. Lumbar and Cervical Spine (facet joint OA)
Joint Injury and Post-Traumatic Osteoarthritis1. Knee Meniscal tears in setting of:
a. traumatic knee injury
b. idiopathic
2. Knee ACL tears
3. Chondral injury/avulsion
4. Hip Labral tears in setting of:
a. traumatic hip injury
b. Femoroacetabular impingement (FAI)
c. idiopathic
Secondary Joint Degeneration/OA in Setting of:1. Anatomic abnormality (i.e. SCFE, FAI w/o labral tear, etc)
2. Muscle Spasticity (i.e. post-CVA, spinal cord injury, Cerebral Palsy, etc.)
3. Deposition diseases:
a. Hemachromatosis
b. Calcium Pyrophosphate Deposition Disease
c. Ochronosis
4. Conditions causing chronic hemarthroses:
a. Hemophilia and other bleeding diatheses
b. anticoagulation therapy
Materials and MethodsDescription of KOOS Outcome Scores:
To assess clinical symptoms and knee disability, The Knee Injury and Osteoarthritis Outcome Score (KOOS) surveys were administered at the time surgery was scheduled to evaluate functional status and symptomatology in the repository cohort. The KOOS is a validated knee-specific patient questionnaire which evaluates both short-term and long-term symptoms and function in patients with knee injury and osteoarthritis. It consists of 5 scored subscales: Pain, other symptoms, Activities of Daily Living (ADL), Function in Sports and Recreation, and Quality of Life (QOL). A normalized score is calculated for each subscale where 100=no symptoms and 0=extreme symptoms.
Enablement of Gene Expression and Innate DB.
Enablement of methods to analyze relative gene expression and to analyze mammalian innate immune responses are in Livak et al. (2001) and Lynn et al. (2008).
These publications are incorporated by reference to the extent they relate materials and methods disclosed herein.
- Livak K and Schmittgen, T., Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔCT Method., Methods 2001:25 402-408
- Lynn D J, Winsor G L, Chan C, Richard N, Laird M R, Barsky A, et al. “InnateDB: facilitating systems-level analyses of the mammalian innate immune response.” Mol Syst Biol 2008; 4:218.
- Lysholm J, Gillquist J. “Evaluation of knee ligament surgery results with special emphasis on use of a scoring scale.” Am J Sports Med 1982; 10(3):150-4.
- Roos E, Toksvig-Larsen S. Knee injury and Osteoarthritis Outcome Score (KOOS)-validation and comparison to the WOMAC in total knee replacement. Health and Quality of Life Outcomes 2003; 1(1):17. http://www.koos.nu/KOOSGuide2003.pdf
- Roos E M, Roos H P, Lohmander L S, Ekdahl C, Beynnon B D. Knee Injury and Osteoarthritis Outcome Score (KOOS)—development of a self-administered outcome measure. J Orthop Sports Phys Ther 1998; 28(2):88-96.
Claims
1. A gene expression profile comprising values for gene products that are differentially expressed in knee injury patients with synovial inflammation compared to knee injury patients without synovial inflammation.
2. The gene expression profile of claim 1 comprising the genes of Annex Table 2.
3. The gene expression profile of claim 2 comprising the genes of Table 3 (SEQ ID NOS 1-65).
4. The gene expression profile of claim 1 wherein the gene products are selected from a group consisting of mRNA and proteins.
5. The gene expression profile of claim 1 wherein cytokine expression is positively associated with Lysholm scores.
6. The gene expression profile of claim 5 wherein higher CCL19 protein levels are associated with worse symptoms.
7. A genetic expression profile used to detect inflammation associated with a joint injury, the gene products obtained from a biological sample from a joint injury, and the profile determined from measuring the gene products of genes in Table 3 (SEQ ID NOS 1-65).
8. A method to target genes in the gene expression profile of a patient, the method comprising:
- (a) determining which gene expression values show the greatest association with synovial inflammation; and
- (b) targeting those genes for developing appropriate therapies.
9. The gene expression profile of claim 8 wherein chemokines IL8 (SEQ ID NO: 5), CCL5 (SEQ ID NO: 3), CCL19 (SEQ ID NO: 1) and CCR7 (SEQ ID NO: 4) are associated with synovial inflammation.
10. A method to treat inflammation associated with knee injuries in a patient, the method comprising:
- (a) determining a gene expression profile of the patient according to claim 1, and identifying genetic targets for therapeutic intervention as those genes within the profile whose expression has the greatest association with synovial inflammation; and
- (b) treating the patent by interacting with the targets to alleviate their effects.
11. The method of claim 10 wherein the targets are cytokines.
12. A method to identify a patient with knee symptoms associated with synovial inflammation, the method comprising:
- (a) determining a gene expression profile from a biological sample of the patient; and
- (b) comparing the profile of the patient to profiles of claim 1 obtained from patients with knee injuries who had synovial inflammation and those who did not, to determine whether synovial inflammation contributes to knee symptoms of the patient.
13. A method to improve clinical outcomes after arthroscopic and post joint trauma in a patient, the method comprising:
- (a) determining the chemokine signature of the patient; and
- (b) developing appropriate treatment based on the target genes that are in the chemokine signature.
Type: Application
Filed: Mar 8, 2013
Publication Date: Jul 11, 2013
Applicant: RUSH UNIVERSITY MEDICAL CENTER (Chicago, IL)
Inventor: RUSH UNIVERSITY MEDICAL CENTER (Chicago, IL)
Application Number: 13/791,484
International Classification: C12Q 1/68 (20060101);